Field of the Invention
The present invention relates to a cell culture medium containing uridine and N-acetyl-D-mannosamine, and a method for production of a glycoprotein comprising culturing cells transformed by an exogenous DNA encoding the glycoprotein by using the cell culture medium.
Discussion of the Background
As a predominant mode of linkage between a protein and a glycan in a glycoprotein, there are N-glycosidic bond where N-acetyl-D-glucosamine is covalently linked to the asparagine residue composing the protein (N-linked glycan, N-glycoside-linked sugar chain), and O-glycosidic bond where N-acetyl-D-galactosamine is covalently linked to the serine or the threonine residue composing the protein (O-linked glycan, O-glycoside-linked sugar chain)
In mammalian cells, N-glycosidic bond-linked sugar chains of glycoproteins are those attached to asparagine residues of the proteins, through a complex pathway involving various enzymes, while the proteins, after translated from RNA, are transferred through the lumen of an endoplasmic reticulum to Golgi bodies. The major types of N-glycosidic bond-linked sugar chains are complex-type sugar chains and high mannose-type sugar chains. Complex-type sugar chains, while there are various types of them, are characterized in that their non-reducing ends consist of sialic acid residues. An example of them is shown in structural formula 2 below.

If mammalian cells, such as Chinese hamster ovary cells (CHO cells), are used in producing a recombinant glycoprotein, a large part of the sugar chains of the protein thus obtained will be a complex-type in their structure, and sialic acid residues thus will occur at the non-reducing ends of the sugar chains of such a recombinant glycoprotein. It is known that stability in the blood of a recombinant protein administered to a body is increased if complex-type sugar chains, which have sialic acid residues at their non-reducing ends, are attached to the protein (cf. Patent Document 1). Thus, when recombinant glycoproteins are to be produced which exhibit their effects while circulating in the blood, production methods using CHO cells are utilized, with which sugar chains are produced having sialic acid residues at their non-reducing ends, expecting elongation of their half-lives in the blood, and thereby augmentation of their pharmacological effects. Erythropoietin and follicle-stimulating hormone (FSH) are typical examples of such recombinant glycoproteins (cf. Patent Documents 2 and 3).
And as a half-life of a glycoprotein in blood is expected to be prolonged when more sialic residues are added to sugar chain of the glycoprotein, several methods are reported objective of which is to added more sialic residues to the glycoprotein. For example, a method is reported for the production of a recombinant glycoprotein by using host cells transformed with sialyltransferase having ability to add a sialic acid to the end of sugar chains (cf. Patent Document 4). Further, reported is that a recombinant glycoprotein added with more sialic acid residues are to be obtained by culturing mammalian cells transformed with a gene encoding a glycoprotein with the temperature lowered in a stepwise manner (cf. Patent Document 5).
Further several cell culture mediums are reported which are to be used for adding more sialic acids to a sugar chain of a glycoprotein. For example, reported is that the molar ratio of sialic acids to a recombinant glycoprotein expressed by cells can be adjusted by adding alkanoic acid such as butylic acid or the salt thereof at a predetermined concentration when the cells cultured (cf. Patent Document 6). And it has been reported that a recombinant glycoprotein having a specific degree of sialylation can be produced by adding a precursor of sialic acid such as ManNAc, acetyl-ManNAc, peracetyl-ManNAc, or fetuin to a medium (cf. Patent Document 7). And it has been reported that sialylation level of a recombinant glycoprotein can be increased by adding monosaccharides, for example, in the combination of glucose, mannose, and galactose, to a medium (cf. Patent Document 8).